Succinic acid (HOOCCH2CH2COOH), a dicarboxylic acid consisting of 4 carbons, is an organic acid having high utilities, which is widely used as a precursor of medicine, food, cosmetics, and chemical products of other industries (Zeikus et al, Appl. Microbiol. Biotechnol., 51:545, 1999; Song et al, Enzyme Microbial Technol., 39:352, 2006). Particularly, the demand for succinic acid is expected to be dramatically increased as a main source of biodegradable macromolecules, with the latest sharp increase in petroleum prices (Willke et al, Appl. Microbiol. Biotechnol., 66:131, 2004).
Succinic acid can be produced by chemical synthesis and fermentation. However, most of succinic acid for industrial use is currently produced through chemical synthesis methods using n-butane and acetylene derived from petroleum as a raw material by Chinese chemical companies, Japanese chemical companies, and big Chemical companies such as BASF, DuPont, BP chemical etc., but only a small amount of succinic acid for special use such as medicine etc. is produced by traditional microbial fermentation method. The above-mentioned chemical synthesis methods have a problem of discharging large amounts of hazardous waste, effluent, and waste gas (e.g., CO, etc.) generated during a process of producing succinic acid. Particularly, fossil fuels having high possibility of being exhausted are used as basic material, and thus there is an urgent need to develop a method for preparing succinic acids to replace the fossil fuels with alternative fuels such as renewable resources.
To overcome these problems caused by the chemical synthesis process for preparing succinic acid, studies on producing succinic acids by microbial fermentation using various renewable resources have been intensively and widely conducted by many researchers. Microorganisms, which have been used in succinic acid production, vary, but they can be generally classified into recombinant Escherichia coli, ruminal bacteria (Actinobacillus, Bacteroides, Mannheimia, Succinimonas, Succinivibrio, etc.) and Anaerobiospirillum (Song et al., Enzyme Microbial Technol., 39:352, 2006).
Among studies on producing succinic acids using recombinant E. coli, there was an attempt to increase succinic acid production by preparing a mutant AFP111 (ATCC No. 202021) obtained through a method in which a glucose transport gene (ptsG) is manipulated while genes (ldh and pfl), which are involved in producing lactic acid and formic acid in E. coli, are eliminated, by the University of Chicago research team (U.S. Pat. No. 5,770,435).
The present inventors have amplified a malic enzyme gene (sfcA) involved in succinic acid production, in recombinant E. coli, NZN111 strain, from which ldh and pfl genes are eliminated, to suppress pyruvic acid accumulated in the fermentation process of the NZN111 strain, thus increasing succinic acid production (Hong et al., Biotechnol. Bioeng., 74:89, 2001). Also, a Georgia University-led team of researchers has constructed an AFP111/pTrc99A-pyc strain by expressing a pyruvate carboxylase gene (pyc) in the AFP111 strain, and then used this strain in producing succinic acid (Vemuri et al, J. Ind. Microbiol. Biotechnol., 28:325, 2001). Recently, in order to induce the production of succinic acid in anaerobic conditions, a Rice University-led team of researchers reported that they have constructed recombinant E. coli strains by manipulating genes involved in pathways of Glycolysis, TCA cycle, and Glyoxylate (Lin et al., Eng., 7:116, 2005; Lin et al., Biotechnol. Bioeng., 90:775, 2005).
Actinobacillus strain and Mannheimia strain, which are a kind of rumen bacteria, and Anaerobiospirillum strain, are known to be excellent in producing succinic acid, so that studies on the strains have been actively conducted. Michigan Biotechnology Institute (MBI)-led team of researchers in America discovered Actinobacillus succinogenes 130Z strain (ATCC No. 55618) to develop a method for producing succinic acid, and constructed various mutant strains of Actinobacillus succinogenes, using traditional chemical mutagenesis to use in developing a process for producing and purifying succinic acid (U.S. Pat. No. 5,521,075; U.S. Pat. No. 5,168,055; U.S. Pat. No. 5,143,834).
However, succinic acid production process using microbial fermentation, developed until now, has a very low productivity of less than 2 g/L/h, and especially it incures a huge cost to separate and purify succinic acid because succinic acid is produced together with large amounts of various organic acids and ethanol as byproducts to some degree during fermentation. Although the above-mentioned results showed an effect of decreasing lactic acid, formic acid, acetic acid, and ethanol as byproducts in some recombinant strains, they did not show complete elimination of them. In addition, in another recombinant mutant strains, there were some cases where the growth rates of them have become so low that overall succinic acid productivity was not increased. Therefore, there is an urgent demand to develop a novel succinic acid-producing strain, which has a high productivity of succinic acid and prevents the production of byproducts (Hong et al., Biotechnol. Lett., 22:871, 2000).
To develop a novel succinic acid-producing strain to satisfy the above demands, isolation of a strain having excellent succinic acid productivity, completion of genome sequence thereof, an understanding of metabolic characteristic thereof, and establishing a genetic manipulation technique required for the construction of a recombinant strain should be preceded. Up to now, in the case of bacteria having high succinic acid productivity, the full genome sequence of strain M. succiniciproducens MBEL 55E (KCTC 0769BP) was completed, but those of bacteria such as Actinobacillus, Anaerobiospirillum etc. have not been reported yet. Although an attempt to try to produce succinic acids by amplifying phosphoenolpyruvate carboxykinase gene (pckA) of A. succinogenes and A. succiniciproducens in E. coli, has been reported (Kim et al., Appl. Environ. Microbiol., 70:1238, 2004; Laivenieks et al., Appl. Environ. Microbiol., 63:2273, 1997), there has been no attempt to try to develop a recombinant succinic acid production strain based on genome sequence.
The present inventors have reported that they isolated M. succiniciproducens MBEL 55E (KCTC0769BP) producing succinic acid with high efficiency from Korean native cattle, and completed genome sequence and characterized metabolic properties of the strain (Hong et al., Nature Biotechnol., 22:1275, 2004). Also, the present inventors have constructed a bacterial mutant, M. succiniciproducens LPK (KCTC10558BP) by disrupting a gene encoding lactate dehydrogenase (ldhA) and a gene encoding pyruvate formate-lyase (pfl) in M. succiniciproducens MBEL 55E (KCTC 0769BP) which is a kind of rumen bacteria in order to inhibit the production of lactic acids and formic acids. In addition to that, the present inventions have constructed a mutant M. succiniciproducens LPK7 (KCTC1062BP) by disrupting a phosphotransacetylase gene (pta) and an acetate kinase gene (ackA) in the mutant strain, M. succiniciproducens LPK in order to inhibit the production of acetic acid, to culture the bacterial mutants in anaerobic conditions (WO 05/052135 A1; Lee et al., Appl. Environ. Microbiol. 72:1939, 2006), thus increasing succinic acid. However, in case of such mutant strains, although the production of byproducts, formic acid and acetic acid could be suppressed to some extent, a large amount of pyruvic acids were accumulated as a byproduct during fermentation, most of all, the growth rate of the strain has become so low compared with a wild strain that an excellent succinic acid productivity could not be achieved.
Meanwhile, it was reported that a pyruvate formate-lyase gene (pfl) participates in conversion of pyruvic acid into acetyl Coenzyme A (acetyl-CoA), thus affecting cell growth and redistribution of pyruvic acid (Wolfe, Microbial. Mol. Biol. Rev., 69:12, 2005).
Accordingly, the present inventors have made extensive efforts to construct a mutant microorganism capable of producing homo-succinic acids at a high yield by minimizing a decrease in microbial growth rate and completely inhibiting the formation of various byproducts including pyruvic acids and to develop a fermentation method thereof, and as a result, they have constructed a bacterial mutant M. succiniciproducens PALK (KCTC10973BP) by disrupting a lactate dehydrogenase gene (ldhA), a phosphotransacetylase gene (pta), and an acetate kinase gene (ackA) without disrupting a pyruvate formate-lyase gene (pfl) in M. succiniciproducens MBEL55E (KCTC 0769BP) which is a kind of rumen bacteria, and then fermentated the mutant strain in anaerobic conditions using glucose and glycerol as carbon sources, and confirmed that the mutant strain can produce nearly homo-succinic acid at a high yield, thereby completing the present invention.